Strychnine affects catecholamine secretion from bovine adrenal medulla chromaffin cells

The effect of strychnine on evoked release of catecholamines from a primary culture of bovine adrenal medullary cells was investigated. Strychnine at > 1 M inhibited catecholamine release stimulated by 10 M acetylcholine, or 10 M nicotine, but not by excess K⁺ (59 mM), the sodium ionophore veratridine (100 M) or the calcium ionophore A-23187 (10 M). The inhibitory response elicited by exposure of the cells to strychnine was rapid (< 3 min) and competitive with acetylcholine. High concentrations of acetylcholine (1 mM) completely overcame this inhibition. Strychnine might be acting on a regulatory site of the nicotinic-cholinergic receptor, which is genetically similar to the strychnine-binding 48 KD subunit of the glycine receptor.     

Inhibitory action of hydralazine on catecholamine secretion from cultured bovine adrenal chromaffin cells

Hydralazine caused a concentration-dependent inhibition of the secretion of catecholamines induced by carbamylcholine or high K+ from cultured bovine adrenal chromaffin cells, and also caused the significant inhibition of radioactive calcium uptake induced by carbamylcholine into the cells. However, hydralazine failed to inhibit the secretion of catecholamines evoked by the calcium-ionophore, A23187. The inhibitory action of hydralazine on catecholamine secretion induced by carbamylcholine was not affected by increasing the concentration of calcium ion in the reaction mixture. These observations therefore seem to indicate that the inhibitory action of hydralazine is not due to either the blocking of receptors for carbamylcholine or the disruption of the secretory machinery, and suggest that the drug may cause the inhibition of catecholamine secretion through its blocking action on calcium influx into the cells.     

Short term effect of steroids on catecholamine secretion from bovine adrenal medulla chromaffin cells

Bovine chromaffin cells were used to examine neuronal modulation, as their function is similar to sympathetic post-ganglionic neurons. The effect of steroids on evoked catecholamine secretion from primary culture of bovine adrenal medullary cells was investigated. A wide range of progestins, androgens and estrogens was found to have a significant effect on catecholamine secretion induced by the natural neurotransmitter acetylcholine (ACh). The androgens (especially androstandione and androsterone), as a class were the most effective in inhibition of stimulated secretion, while the estrogens had little, to no, effect. Among all steroids tested, progesterone had the most significant effect, other progestins were less potent. Progesterone inhibited catecholamine secretion evoked by ACh, nicotine and oxotremorine-M in a dose-dependent manner with similar IC₅₀ values in the μM range. It also blocked the secretion evoked by high potassium concentration (59 nM) or veratradine (100 μM), but no effect was seen on the secretion evoked by the calcium ionophore A-23187 (10 μM). Progesterone inhibition of ACh or oxotremorine-M stimulation was immediate and sustained. These results suggest that progesterone and other steroids might have a membrane effect probably acting through blockade of calcium influx necessary for the secretory response.     

The effects of neosurugatoxin on evoked catecholamine secretion from bovine adrenal chromaffin cells.

1. The effect of neosurugatoxin (NSTX), a toxin from the Japanese ivory mollusc (Babylonia japonica), on the nicotinic response of bovine adrenal chromaffin cells was examined. 2. NSTX inhibited acetylcholine- and nicotine-induced catecholamine secretion from the cultured cells with an IC50 against 5 microM nicotine of 30 nM. 3. This inhibitory effect was reversible and independent of the presence of agonist. 4. NSTX had no effect on the catecholamine release from cultured cells evoked by 50 mM K+, or 1 microM histamine. 5. NSTX had no effect on the stimulation of phosphatidylinositol metabolism evoked by 100 microM muscarine. 6. These results suggest NSTX may be useful as a nicotinic receptor probe in tissues such as the adrenal and sympathetic ganglia where alpha-bungarotoxin is ineffective.     

Interferon-α reduces catecholamine secretion from bovine adrenal chromaffin cells stimulated by acetylcholine

A long-term pretreatment (72h) of bovine adrenal chromaffin cells with recombinant human interferon (IFN)-α-2b (1500 units/ml) produced a decrease in the secretion of catecholamines from the cells stimulated by acetylcholine (ACh) (25μmol/l) but not that with human fibloblast IFN-β (3000 units/ml) or recombinant human IFN-γ (3000 units/ml). IFN-α-2b inhibited the ACh-induced secretion in a concentration- (30–1500 units/ml) and time-dependent manner (18–72h). The content of catecholamines in the cells treated with IFN-α-2b for 72h did not change. The inhibitory effect of IFN-α-2b on the secretion was abolished when the cells were simultaneously treated with anti-IFN-α antibody, and it was overcome by the increase in the external ACh concentration. IFN-α-2b also inhibited ACh-induced Ca²⁺ influx into the cells in a concentration-dependent manner similar to that of the IFN-α-2b inhibiting ACh-induced secretion. On the other hand, IFN-α-2b failed to reduce the secretion from the cells induced by high K⁺. These results strongly suggest that IFN-α-2b reduces the ACh-induced secretion of catecholamines from bovine adrenal chromaffin cells due to modulating the gene expression of the nicotinic ACh receptor-operated cation channels rather than due to directly affecting the channels. The results further indicate that the IFN-α-2b inhibition may be associated with the psychiatric side effects of IFN-α (depression, neurasthenica and somnolence, etc.), and that immune systems may regulate the function of (autonomic) nervous systems or adrenal medulla via IFN-α in vivo. Received: 6 March 1997 / Accepted: 31 July 1997     

Effects of extract and ingredients isolated from Magnolia obovata thunberg on catecholamine secretion from bovine adrenal chromaffin cells

The crude extract of magnolia bark, an herbal drug, inhibited the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine (ACh) in a concentration-dependent manner (200-900 microg/mL). The extract also diminished the secretion induced by high K(+), which is a stimulus directly depolarizing the plasma membranes, but its inhibition was weaker than that of ACh-evoked secretion. beta-Eudesmol, honokiol, magnolol, and bornyl acetate, but not alpha- and beta-pinenes, all of which are ingredients of magnolia bark, greatly reduced ACh-evoked secretion. beta-Eudesmol and magnolol also inhibited high K(+)-induced secretion to an extent similar to that of ACh-evoked secretion. However, honokiol and bornyl acetate inhibited the secretion induced by high K(+) much less than the secretion evoked by ACh. ACh-induced Na(+) influx and ACh- or high K(+)-induced Ca(2+) influx into the cells were diminished by beta-eudesmol or honokiol. These results indicate that magnolia bark contains some effective components inhibiting the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by ACh due to the antagonism of Na(+) and Ca(2+) influxes into the cells. However, inhibition by the extract of magnolia bark seems to be attributable to honokiol and bornyl acetate. Furthermore, the results indicate that the inhibitory effect of magnolia bark may be associated with its pharmacological effect on activities of the nervous system.     

Effects of deltamethrin on catecholamine secretion of bovine chromaffin cells

The effects of the pyrethroid deltamethrin (D) on catecholamine secretion of cultured bovine chromaffin cells were investigated in vitro using high performance liquid chromatography (HPLC). Spontaneous release of catecholamines was increased by 10 μM and 100 μM D. This increase could partially be prevented by the simultaneous use of 2 μM tetrodotoxin (TTX), which reduced the increase by 10 μM D of catecholamine secretion by 90% and that of 100 μM D by 50%. TTX 2 μM alone did not alter the spontaneous release in comparison to controls. Medullary chromaffin cells consist of two cell groups, one secreting mainly epinephrine (E), the other norepinephrine (NE). The ratio between the spontaneously secreted catecholamines E and NE was increased after treatment with D, indicating a dominant effect on E secreting cells. Received: 7 March 1994/ Accepted: 26 April 1994     

Inhibition of nicotinic receptor-mediated catecholamine secretion by dryobalanops aromatica in bovine adrenal chromaffin cells

Effect of the aqueous extract from a medicinal plant Dryobalanops aromatica(Dipterocarpaceae) on catecholamine secretion was investigated in bovine adrenal chromaffin cells. The aqueous extract inhibited [(3)H]norepinephrine ([(3)H]NE) secretion induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), a nicotinic acetylcholine receptor (nAChR) agonist, with a half-maximal inhibitory concentration (IC(50)) of 8.4 +/- 1.7 microgml(-1). Increases in cytosolic calcium ([Ca(2+)](i)) and sodium ([Na(+)](i)) induced by DMPP were also inhibited by the extract. However, the binding of [(3)H]nicotine to nAChRs was not affected by the addition of the extract in receptor binding competition analysis, suggesting that active components in the extract and nicotine do not share the binding site in the nAChR. On the other hand, [Ca(2+)](i)increases induced by high K(+), ionomycin, bradykinin, angiotensin II, and thapsigargin were not inhibited by the extract. The data suggest that the extract from D. aromatica specifically inhibits catecholamine secretion by blocking nAChR in a noncompetitive manner.     

Effects of opioid peptides and morphine on histamine-induced catecholamine secretion from cultured, bovine adrenal chromaffin cells.

The effect of opioid peptides and morphine on histamine-induced catecholamine secretion has been studied in monolayer cultures of dispersed, bovine adrenal chromaffin cells. Histamine-induced a dose-dependent secretion of both adrenaline and noradrenaline with a threshold dose of approximately 5 nM, an EC50 of 150 nM and maximal secretion at 10 microM. Catecholamine secretion induced by 1 microM histamine was completely dependent on extracellular calcium, was inhibited in a dose-dependent manner by mepyramine (1 nM-1 microM), and was unaffected by cimetidine (10 microM) and hexamethonium (0.1 mM). Dynorphin-1-13 (1 nM-20 microM), metorphamide (0.1 nM-10 microM), morphine (1 nM-0.1 mM) and diprenorphine (1 nM-0.1 mM) each had no effect on adrenaline or noradrenaline secretion induced by 1 microM histamine. The characteristics of histamine-induced catecholamine secretion from bovine adrenal chromaffin cells were similar to those reported previously for cat and rat adrenal medulla being calcium-dependent and mediated by H1 histamine-receptors. The results with opioid peptides and morphine suggest that endogenous adrenal opioid peptides do not act on the opioid binding sites found on adrenal medullary chromaffin cells to modify their secretory response to histamine.     

Inhibitory action of novel arginine derivative on catecholamine secretion evoked by acetylcholine from cultured bovine adrenal chromaffin cells

A novel product, 4-amino-5-guanidinopentanoic acid 15-[(4-aminobutyl)-3-aminopropylcarbamoyl] pentadecyl ester (Arg-HSA-Spm), was synthesized based on ptilomycalin A, which is one of the extracts from marine sponge. Arg-HSA-Spm contains arginine in its chemical structure. The pharmacological action of Arg-HSA-Spm on catecholamine secretion from cultured bovine adrenal chromaffin cells was examined. Arg-HSA-Spm inhibited catecholamine secretion stimulated by the physiological secretagog acetylcholine. This inhibitory action of Arg-HSA-Spm on catecholamine secretion induced by 10(-4) M acetylcholine was dose-dependent from 10(-8) M to 10(-5) M. In the presence of 3 x 10(-7) M Arg-HSA-Spm, the stimulation of catecholamine secretion observed by increasing acetylcholine up to 10(-3) M did not reach the maximal level observed without Arg-HSA-Spm. Arg-HSA-Spm at 10(-5) M suppressed both the increase in intracellular free Ca2+ level and the influx of 45Ca2+ induced by 10(-4) M acetylcholine. The Arg-HSA-Spm-induced suppression of intracellular free Ca2+ level, the influx of 45Ca2+ and catecholamine secretion were not observed in the presence of extracellular K+ at 56 mM. The results presented in this study suggested that Arg-HSA-Spm may inhibit the influx of extracellular Ca2+ into the cells, probably through its blocking action related to acetylcholine receptors, resulting in the inhibition of catecholamine secretion in adrenal chromaffin cells.     

Role of Ca2+/phospholipid-dependent protein kinase in catecholamine secretion from bovine adrenal medullary chromaffin cells

The role of Ca2+/phospholipid-dependent protein kinase (protein kinase C) in catecholamine secretion from bovine adrenal medullary chromaffin cells was examined using four protein kinase C inhibitors: polymyxin B, sphingosine, staurosporine, and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). For this purpose, digitonin-permeabilized chromaffin cells were used. Secretion of catecholamines from these cells was stimulated by the addition of micromolar amounts of exogenous free Ca2+. 12-O-Tetradecanoylphorbol-13-acetate (TPA) and arachidonic acid, activators of protein kinase C, enhanced the catecholamine secretion evoked by Ca2+. But phorbol-12, 13-diacetate, a phorbol ester analog that does not activate protein kinase C, had no effect on Ca2(+)-evoked secretion. Polymyxin B at a low concentration (1 microM) abolished the enhancement of secretion by TPA or arachidonic acid without affecting the secretion evoked by Ca2+. However, polymyxin B at higher concentrations (10-100 microM) greatly reduced Ca2+-evoked catecholamine secretion. Sphingosine 10 microM-1 mM), Staurosporine (100 nM-1 microM, and H-7 (100-500 microM) inhibited TPA- or arachidonic acid-enhanced secretion but not Ca2(+)-evoked secretion. In cells in which protein kinase C was down-regulated by TPA, specific binding of [3H]phorbol-12,13-dibutyrate to the cells almost disappeared and the enhancement of secretion by TPA was no longer observed, whereas Ca2(+)-evoked secretion was maintained. These results strongly suggest that protein kinase C is not essential for the Ca2(+)-dependent catecholamine secretion from bovine adrenal chromaffin cells, but acts instead as a modulator.     

Characterization of ginseng saponin ginsenoside-Rg(3) inhibition of catecholamine secretion in bovine adrenal chromaffin cells.

Since ginsenoside-Rg(3), one of the panaxadiol saponins isolated from the ginseng root, significantly inhibited the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine (ACh), the properties of ginsenoside-Rg(3) inhibition were investigated. Although ginsenoside-Rg(3) inhibited the secretion evoked by ACh in a concentration-dependent manner, it affected the secretion stimulated by high K(+) or veratridine, an activator of the voltage-sensitive Ca(2+) or Na(+) channels, only slightly. The ACh-induced Na(+) and Ca(2+) influxes into the cells were also reduced by ginsenoside-Rg(3). The inhibitory effect of this saponin on the secretion of catecholamines was not altered by increasing the external concentration of ACh or Ca(2+). The ACh-evoked secretion of catecholamines was completely restored in cells that were preincubated with 10 microM ginsenoside-Rg(3) and then incubated without the saponin, whereas secretion was not completely restored in cells that were preincubated with 30 microM of this compound. Above 30 microM ginsenoside-Rg(3) increased the fluorescence anisotropy of diphenylhexatriene in the cells. Furthermore, the inhibitory effect of ginsenoside-Rg(3) at 30 microM on the ACh-evoked secretion of catecholamines was dependent upon the preincubation time, but this was not the case at 10 microM. These results strongly suggest that ginsenoside-Rg(3) blocks the nicotinic ACh receptor-operated cation channels, inhibits Na(+) influx through the channels, and consequently reduces both Ca(2+) influx and catecholamine secretion in bovine adrenal chromaffin cells. In addition to this action, the ginsenoside at higher concentrations modulates the fluidity of the plasma membrane, which probably contributes to the observed reduction in the secretion of catecholamines.     

Prostaglandin E2-induced arachidonic acid release and catecholamine secretion from cultured bovine adrenal chromaffin cells.

We recently reported that prostaglandin E2 (PGE2) and arachidonic acid (AA) each induced a gradual secretion of catecholamines from cultured bovine adrenal chromaffin cells in the presence of ouabain by stimulation of phosphoinositide metabolism. In the present study, we examined the relationship between phospholipase A2 and C activation and catecholamine secretion by PGE2 in chromaffin cells. The phospholipase A2 inhibitors p-bromophenacyl bromide and mepacrine did not affect the basal and ouabain-induced release, but dose-dependently blocked PGE2-evoked phosphoinositide metabolism and the consequent catecholamine release at an IC50 value of 3 microM. PGE2 induced rapid hydrolysis of [3H]AA from prelabeled phospholipid pools: the release of [3H]AA could be detected at as early as 15 sec and reached a plateau after 1 min. While the phospholipase C inhibitor neomycin did not inhibit PGE2-induced AA release, phospholipase A2 inhibitors dose-dependently inhibited it at IC50 values comparable to those for catecholamine release. Pretreatment of intact cells with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, but not with pertussis toxin, prevented AA release by PGE2. These results demonstrate that PGE2 activates phospholipase A2 as well as phospholipase C in a pertussis toxin-insensitive manner and suggest that the released arachidonic acid may be involved in PGE2-induced catecholamine release from chromaffin cells.     

Inhibitory action of cilostazol,a phosphodiesterase III inhibitor,on catecholamine secretion from cultured bovine adrenal chromaffin cells

The effect of cilostazol, a phosphodiesterase III inhibitor, on catecholamine secretion was examined using bovine adrenal chromaffin cells in culture. Catecholamine secretion evoked by acetylcholine was markedly inhibited by cilostazol. In contrast, 56 mM K+-evoked secretion was slightly inhibited by cilostazol. Cilostazol elevated the level of cyclic AMP in the cells. However, a cyclic AMP analog (dibutyryl cAMP) or an adenylate cyclase activator (forskolin) failed to inhibit secretion of catecholamine induced by acetylcholine. Cilostazol decreased the level of intracellular free Ca2+ stimulated by acetylcholine. Cilostazol thus inhibits secretion of catecholamine through its blocking action on Ca2+ movement in the adrenal chromaffin cells.     

Inhibition and facilitation by pimobendan,a calcium sensitizer,of catecholamine secretion from bovine adrenal chromaffin cells

The effects of pimobendan, a Ca(2+) sensitizer with inhibitory action against cyclic-GMP-inhibited phosphodiesterase (PDE-III), on catecholamine (CA) secretion were studied in bovine adrenal chromaffin cells. In intact cells, pimobendan (10 - 100 microM) inhibited CA secretion stimulated by acetylcholine (10 and 30 microM) and 1,1-dimethyl-4-phenyl-piperazinium (DMPP) (3 and 10 microM), but facilitated CA secretion stimulated by high K(+) (30 mM), histamine (3 microM), and angiotensin-II (3 microM). Histamine and angiotensin-II had no effect on CA secretion in Ca(2+)-free medium. The inhibition or facilitation by pimobendan of the stimulation-evoked CA secretion was not affected by H-89 (1 microM) and H-8 (30 microM), inhibitors of cyclic-AMP-dependent protein kinase. Milrinone (10 and 30 microM) and amrinone (100 and 300 microM), inhibitors of PDE-III, did not affect the stimulation-evoked CA secretion. In beta-escin-permeabilized cells, pimobendan (10 - 100 microM) did not affect CA secretion stimulated by Ca(2+) (0.1 - 10 microM) in the presence and absence of MgATP (2 mM). These results indicate that pimobendan has dual effects, inhibition and facilitation, on CA secretion. The inhibition may be due to an inhibitory action on nicotinic receptors and the facilitation may be due to a facilitatory action on stimulation-induced Ca(2+) influx. Neither Ca(2+) sensitizing nor PDE-III inhibiting actions seem to be related to these effects.     

Multiple calcium channels are required for pituitary adenylate cyclase-activating polypeptide-induced catecholamine secretion from bovine cultured adrenal chromaffin cells

The effects of L-, N-, P- and Q-type calcium channel antagonists and (±)-BayK-8644 on catecholamine release induced by pituitary adenylate cyclase-activating polypeptide (PACAP-27) were investigated in bovine cultured adrenal chromaffin cells. PACAP-27 induced the release of 4-15% of the total cellular catecholamines over 7 min, with an EC₅₀ of 20 nM and the effect approaching maximum at 100 nM. Catecholamine release was fully dependent on the presence of extracellular calcium. The dihydropyridine nitrendipine which inhibits L-type calcium channels inhibited PACAP-27-induced secretion in a concentration dependent manner with an inhibition of 20-30% at 1 μM. In contrast, (±)-BayK-8644, which prolongs the opening of L-type calcium channels produced a concentration-dependent increase in PACAP-27-induced catecholamine release with 1 μM increasing release by 40-60%. Blockade of N-type calcium channels with ω-conotoxin GVIA reduced release by 5–15%. Block of P-type channels with low concentrations of ω-agatoxin IVA (≤ 30 nM) had no significant effect on release, while higher concentrations (100-300 nM) which block Q-type channels reduced release by up to 15%. ω-Conotoxin MVIIC, an antagonist of Q-type calcium channels and also of N- and P-type channels, inhibited release in a concentration-dependent manner with a near maximum effect of 30-50% produced by 300 nM. The combination of ω-conotoxin GVIA and ω-agatoxin IVA reduced release by 40-50%. Addition of ω-conotoxin MVIIC (300 nM) to the combination of ω-conotoxin GVIA (10 nM) and ω-agatoxin IVA (100 nM) did not inhibit catecholamine release more than with ω-conotoxin GVIA and ω-agatoxin IVA alone, indicating that 100 nM ω-agatoxin IVA was sufficient to block the Q-type calcium channels. When nitrendipine was used together with ω-conotoxin GVIA, ω-agatoxin IVA and ω-conotoxin MVIIC, catecholamine release induced by 20 nM or 100 nM PACAP-27 was reduced by 70–85%. Taken together these results suggest that influx of calcium through multiple different voltage-sensitive calcium channels mediate PACAP-27-induced catecholamine release from bovine chromaffin cells, and that L-, N- and Q-channels contribute to this response. Received: 11 March / Accepted: 20 June 1997     

Possible muscarinic regulation of catecholamine secretion mediated by cyclic GMP in isolated bovine adrenal chromaffin cells

Catecholamine secretion and cyclic GMP levels were measured in chromaffin cells isolated from bovine adrenal medulla. Acetylcholine (ACh) and nicotine, but not muscarine, induced 8- to 10-fold increases in catecholamine secretion, with respective ED₅₀ values of 10 and 2 M. Cyclic GMP levels were also increased from 3- to 5-fold in the presence of ACh, and this stimulation was mimicked by muscarine but not by nicotine. Half-maximum stimulations of cyclic GMP levels with ACh and muscarine were observed at 0.1 and 0.3 M respectively. The order of potency of various cholinergic drugs for cyclic GMP stimulation was as follows: ACh > oxotremorine > methacholine > muscarine > carbamylcholine > furthretonium > arecholine > bethanechol. Pilocarpine, McN-A-343, and AHR-602 were inactive at concentrations between 10^?8 and 10^?3 M. Isobutylmethylxanthine (1 mM), a specific phosphodiesterase inhibitor, caused a 7-fold increase in cyclic GMP and potentiated 3-fold the stimulation of cyclic GMP by ACh. The nicotine-induced catecholamine secretion was inhibited 19 and 33 per cent by the co-stimulation of the muscarinic receptor with 0.2 and 0.5 M ACh, respectively. Isobutylmethylxanthine (1 mM) also caused a 44 per cent inhibition of nicotine-induced catecholamine secretion, and its effect was additive to that of ACh. Atropine (0.1 M) selectively abolished the inhibition caused by ACh. Similar inhibitions were also obtained in the presence of exogenous dibutyryl cyclic GMP or 8-bromo cyclic GMP. These data indicate that the nicotinic stimulation of catecholamine secretion from bovine adrenal chromaffin cells may be regulated by cyclic GMP via the stimulation of a muscarinic receptor.